In developing processes of pharmaceuticals, instead of animal testing, in vitro assay using cells is desired. Particularly, it is applied more actively to screening, toxicity and metabolic testing of drug candidate materials.
Against such a background, replacing conventional animal testing, alternative approaches using cells have been tried actively, but many of them have limited capacity to predict clinical reactions. It is considered to be because, in these culture methods, cells do not have structures imitating that of the real-life systems (Non Patent Literature 1). Therefore, construction of three-dimensional tissues which fulfill functions nearer to that of real-life systems have been tried, and three-dimensional tissues of various cells have been successfully made.
As a substrate to form three-dimensional tissues of cells, a sheet for culturing on which extremely microscopic and uniform protrusions are regularly arranged (nanopillar sheet) has been developed, but it has a problem that three-dimensional tissues that have been formed have high peeling property from substrates (Patent Literature 1), and are lost during medium replacement. Also, since diameters of formed three-dimensional tissues cannot be controlled, it has a problem that the diameters are not uniform, and functions of each three-dimensional tissue vary. It remains immature for practical forming.
Accordingly, a technique to provide minute cavity structures on a culture substrate, and to form a three-dimensional tissue per the cavity (with a cellular organization micro chip) has been developed (Patent Literature 2, Non Patent Literature 2). As characters of the technique, by applying adhesive materials to the predetermined regions around the center of bottom surfaces of cavities, cell-adhered regions and cell-not-adhered regions are specified, and by rotating the cavity itself with such as a rotation drive apparatus and carrying out rotational culture, culture cells are held to near the center of bottom surfaces of cavities, cell-adhered regions.
In addition, the present inventor et al., with the aim of spheroid formation having uniform diameters, are carrying out studies of the nanopillar culture sheet (Non Patent Literature 3). The nanopillar culture sheet, by making the surface of the substrate to which cells are adhered a concavo-convex structure, is intended to control diameters of spheroids to be formed.